This invention relates to prostate specific antigen (PSA) and its protein complexes as may be found in blood serum, ascites, tissues, tumors and seminal fluid.
Prostate cancer is one of the most frequently diagnosed cancers among U.S. men, and is the second most common male cancer and is a leading cause of male cancer related mortality. In the United States over 35,000 men die annually from prostate cancer. Since its discovery, prostate specific antigen (PSA) has become the most valuable tool for the diagnosis and management of prostate cancer.
PSA, a single chain glycoprotein of approximately 30 kDa, is a member of the human kallikrein gene family, which consists of hKLK1, hKLK2 and hKLK3 (PSA). All three genes are clustered within 60-kb genome region on chromosome 19 Q13.3-q 13.4. The PSA gene has five exons and encodes a 237 amino acid mature protein which is secreted. PSA protein is glycosylated at a single site (asparagine 45). Each PSA molecule has six immunoreactive binding sites.
PSA is primarily produced by prostate epithelial cells and is secreted into seminal fluid to a high concentration. PSA at low concentrations has been found in endometrium, normal brain tissue, breast tumors, breast milk, adrenal neoplasm and renal cell carcinoma. PSA circulates in the serum as uncomplexed or free form and complexed or bound form. In the serum most of the PSA is complexed with xcex11-antichymotrypsin (ACT) (1,2); and xcex12-macroglobulin (A2M) (3). A small portion of PSA is bound to xcex11-protease inhibitor (API). A complex between PSA and protein C-inhibitor (PCI) is present only in seminal plasma.
Despite the wide spread acceptance and use of serum PSA as a marker, for the early detection of prostate cancer, the specificity of this PSA test is relatively low. The yield of cancer in a screening population is only 22-30%; which means that 70-80% of all test results, indicating that a biopsy should be performed, are false positives. Progressively rising PSA levels above the xe2x80x9cnormal rangexe2x80x9d of 0-4 ng PSA/ml are one of the earliest signs of prostate cancer. As a regular screening for prostate cancer becomes the standard of care, improved specificity in detection of prostate cancer is needed to avoid costly, unnecessary biopsies. PSA is produced by malignant as well as by non-malignant prostate epithelial cells. Therefore, there is a substantial overlap in total PSA levels between men with prostate cancer, benign prostatic hyperplasia (BPH) and chronic prostatitis. Recently, the measurement of the ratio between total PSA and free PSA has been introduced as a useful clinical tool for the early detection of localized prostate cancer in order to increase reliability of the test.
Different commercial PSA immunoassays usually give different results in the same patients. This underscores the need to standardize PSA assays. It is particularly important that PSA immunoassays be standardized in the range of 0-10 ng PSA/ml, because this is the most critical range for a prostate cancer screening program.
The process of standardization of the PSA-immunoassays requires several steps: 1) a standard method for PSA isolation in its native forms must be defined, 2) a method to preserve native PSA for a reasonable period and 3) a serum based PSA standard is needed. This is essential because most of the clinical decisions for patient care are based upon measurement of serum PSA levels whereas most, if not all, PSA standards currently used are from a seminal plasma source. Additionally, recent data indicate that analytical characteristics of seminal fluid PSA differs from that of serum PSA. Current PSA immunoassays are designed to measure total PSA in the serum. To evaluate differences in such assays, one should use xe2x80x9cserum standardsxe2x80x9d containing different proportions of free and complexed PSA and not free PSA obtained from seminal plasma. It has been proposed that only the complexed forms of PSA be used as the internal antigen calibrator for PSA immunoassays. However, at present, no such standards are available.
Current PSA quantitation methodology estimates either free PSA, total PSA or PSA that complexes with xcex11 antichymotrypsin (PSA-ACT). It, however, does not include other known complexes such as xcex12 macroglobulin complex with PSA (PSA-A2M); xcex11-protease inhibitor complex with PSA (PSA-API) and a complex between PSA and protein C inhibitor present only in seminal fluid (PSA-PCI). This is the reason why total PSA determination is often higher than sum total of free PSA and PSA-ACT complex. In order to correctly define the role of different molecular forms of PSA in immunoassays it is essential that an entire panel of PSA molecular forms (free and complexed forms) found in the patient samples, e.g., serum, be represented in standardization of PSA assays and in preparation of a PSA calibrator.
All currently available methods for the quantitation of PSA involve use of monoclonal and polyclonal antibodies to measure free PSA and PSA-ACT complex, usually in the serum. Regardless of the assay procedure used, the value of total PSA always exceeds the sum of free PSA and PSA-ACT complex by up to 30%. Possible reasons for this discrepancy may be: 1) presence of other known PSA-complexes (PSA-A2M, PSA-API), 2) other unknown PSA-complexes, 3) differences in specificity of anti-PSA antibodies-as they are prepared against free PSA from seminal plasma and may have different affinity for free PSA than PSA-complexes, 4) differences in the degradation rate of different PSA molecular forms, 5) use of seminal plasma based PSA as an internal antigen calibrator, 6) use of solid phase for immobilizing the capture antibody which may affect the kinetics of antigen-antibody interaction and 7) any combination of these.
For the most part, the source for the isolation of prostate specific antigen (PSA) for clinical and laboratory use has been seminal plasma and rarely from cultured prostate tumor cells. As of today, all internal standards used for monitoring PSA in patient serum are prepared from seminal plasma. However, the biochemical nature of PSA from seminal plasma and from patient blood may not be identical. Thus far no one has purified PSA or any of its molecular forms directly from the patient serum. Most identification of PSA in patient serum is based upon immunological detection and quantitation using monoclonal and polyclonal antibodies. The validity and accuracy of such measurements largely depends upon the quality and nature of each antibody used. There is no single antibody known today that can capture free PSA and all of its molecular forms.
Thiophilic adsorption chromatography was first introduced by Porath and coworkers in 1985 employing a thiophilic gel containing a sulfone group and at least one thioether function. The synthesis of the gel consisted of coupling 2-mercaptoethanol to agarose that has been activated by divinylsulfone. The adsorption of proteins to the stationary phase of their particular gel and many others is promoted by high concentration of lytropic-salts such as sodium, potassium and ammonium sulfates, whereas desorption is achieved by decreasing salt concentration. Thiophilic adsorption chromatography has been largely used for the purification of immunoglobulins of different classes: IgA, IgG and IgM from different species and for purification of polyclonal antibodies from serum and monoclonal antibodies from tissue cellular supernatants and from ascitic fluid. Ion exchange effects are excluded in view of high salt concentration in the adsorption step. There has, however, been no recorded indication or suggestion that thiophilic gels might have applicability to isolation or purification of PSA.
Thiophilic gels have pendant surface ligands attached to a hydrophilic solid support, e.g. agarose or polyacryamide. The surface structures are ligands containing hydrophilic electron donor and acceptor groups. Commercially there are three different thiophilic gels that contain one, two and three sulfur containing groups respectively. 
The adsorption of proteins to T-gels is usually promoted by xe2x80x9cstructure formingxe2x80x9d (lyotropic) salts. Typically, protein solutions are applied on T-gels at high salt, e.g. sulfate, concentration, about 0.5 to about 1.0 Molar, at about pH 6 to about pH 8, and adsorbed proteins are eluted by a decrease of salt concentration in the mobile phase.
In accordance with the invention, it is therefore an object to provide a method for the isolation of PSA and its molecular forms, e.g. complexes or isoforms, from biological fluids.
It is a further object to isolate PSA and its molecular forms from biological fluids without using monoclonal or polyclonal antibodies.
It is a further object of the present invention to provide a method for isolation of PSA and its molecular forms that provides consistency in quantitative analysis of free PSA, complexed PSA and total PSA.
It is a further object of the present invention to provide a method to separate and quantify free PSA and its various molecular forms and to be able to use the ratios and quantities of free PSA and its various complexes to increase reliability of screening tests for prostate cancer.
It is a further object of the present invention to prepare PSA test standards based upon serum rather than upon seminal fluid as a source of PSA.
The foregoing and other objects of the invention are met by the discovery of a method for capturing and isolating PSA and all of its known complexes from biological fluids using thiophilic gels. Such captured and isolated PSA and PSA complexes can be then quantified by known methods.
The invention includes a method for capturing PSA and its molecular forms that may be in a fluid biological material including the steps of:
a) preparing a chromatographic column by placing a thiophilic gel in a column where the thiophilic gel is formed from a water insoluble polymer where the surface of the gel is provided with thiophilic moieties that bind PSA in the presence of an adsorption liquid but that will release PSA upon elution with an eluting liquid, said thiophilic moieties comprising a two part structure wherein one part can be characterized as a hydrophilic electron acceptor and the other part is sulfur which acts as an electron donor;
b) selecting a sample of a fluid biological material to be tested for PSA and its complexes;
c) introducing the sample into the column;
d) passing the sample through the column;
e) rinsing the column with adsorption liquid to remove materials that are unbound to the thiophilic gel;
f) eluting the column with a liquid that will displace PSA; and
g) capturing PSA, including its complexes, in eluted column fractions.
More particularly, in a preferred embodiment, the method for capturing PSA including its complexes from fluid biological material includes the steps of:
a) preparing a chromatographic column by placing a thiophilic gel in a column where the thiophilic gel is selected from the group consisting of PyS, 2S and 3S;
b) equilibrating the column with an aqueous alkali metal sulfate salt solution at a concentration of from about 0.5 to about 1.0 Molar;
c) selecting a sample of a fluid biological material to be tested for PSA and its complexes;
d) adding alkali metal sulfate to the sample to obtain a salt concentration of from about 0.5 to about 1.0 Molar;
e) introducing the sample into the column;
f) eluting the column with an aqueous alkali metal sulfate salt solution at an original concentration of from about 1.0 molar to 0.5 Molar to remove materials that are not bound to the gel;
g) displacing proteins bound to the gel by rinsing the column with aliquots of aqueous solutions of alkali metal sulfate salt at concentrations incrementally reduced from the about 0.5 to about 0.1 original concentration to obtain column fractions; and
h) analyzing the displaced proteins for PSA.
The invention further includes separation of PSA and its complexes by collecting column fractions and quantification of PSA or PSA complex in the fractions.